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PARASITOLOGY METHODS- OVERSTREET (2007)

Suggested Preparation of Parasites for Light Microscopy Parasites and tissues suspected of hosting parasites or containing a pathological alteration require proper fixation and preparation. This procedure may differ depending on anticipated procedures to be followed in the future or on what materials are available at the time of collection. In any case, the collector should keep in mind that attention to the quality of preservation at this time will save considerable time and effort in the future and may make the difference between exquisite end products and useless trash. The following suggestions work for many specific groups and are good general procedures. They are not necessarily the best, and the reader should feel free to use other methods or compare these with other methods. Blood Parasites Prepare a thin film of blood by placing a small drop of blood near one end of a clean microscope slide and carrying that drop (not pushing it!!) across. To do this, place a second slide at the anterior border of the drop and use that slide to draw the drop. The moving slide should be held at about a 40 angle and swept in a smooth rapid stroke. The smears are left to dry and do not have to be stained immediately. Giemsas stain is usually the best stain to use. Wrights stain is often acceptable, is faster, and contains the fixative. Preparations to be stained in Giemsa should be fixed in absolute methanol for 1-5 minutes, unless slides are going to be used to detect a few parasites occurring in a thick film. In the latter case, dehemoglobinize the preparation in distilled water for 3 minutes after staining. Staining time varies between 30 to 60 (usually 45 to 50) minutes and about 1 drop of the stock solution is used per ml of pH 7.0 7.2 distilled water or buffered water. The same buffered solution is used to wash the slide after being stained in a staining jar. Coverslips may be used if desired. If Wrights stain is used, place enough stain on the dried film to completely immerse the area but not run over the sides of the slide in a slide-rack and let the preparation stand for 3 to 6 minutes. Then add to the stain neutral distilled or buffered water drop by drop until a metallic sheen appears on the meniscus and allow it to remain for about twice the time used for staining. The solution should not run off the slide, and it should immerse the film. Flush the solution off with neutral water until color does not run from the preparation. Drain off the water and dry rapidly. Protozoa other than blood parasites Lugols solution can be used to observe flagella, cilia, and glycogen bodies in fresh preparations. Permanent preparations usually are made by smearing parasitic material thinly on a coverslip. The albuminous substance from the host or media will usually allow the specimens to adhere. Fresh egg-white emulsified in distilled water (or a commercial product) applied in a thin layer on the coverslip (or the slide) might be necessary for the material to stick properly. Let the smear sit 5-10 minutes before fixing. Schaudinns fixative used to be a commonly used fixer. Bouins solution or AFA are also used, especially if certain stains such as Protargols stain are going to be used. Float the coverslip face down in the fixative for about a minute, then reverse the coverslip and submerge same for about 10 minutes. Material should be stained as soon as possible for best results, although coverslips can be stored in fixative or 70% ethyl alcohol. Amoebas can be fixed in polyvinylalcohol (PVA). Place a drop of PVA on

2 a microscope slide and distribute amoebas into the drop and let it dry. These slides may be stored as they are without causing damage to parasites until they are stained, preferably with Mallorys trichrome stain. Heidenhains hematoxylin is recommended for coverslip smears as long as the smears are thin and even. Trematodes (Digeneans) Remove living flukes from their host tissue in a small amount of saline under a dissecting scope by pressing the hosts epithelium below the worms suckers with a pair of fine tipped forceps and lifting the worm from the host tissue and placing it in a small dish (e.g.,Stender dish) containing a portion (usually one quarter the volume or less) of approximately 0.8% saline (or NaCl) to clean the mucus and debris from it. A solution of 0.75% works well for parasites in most fishes, and 0.85% works better if collecting parasites from warm-blooded vertebrates. With practice, removing worms from tissue or saline becomes easy. The worm should not be pinched in any degree with forceps. If worms occur loose in saline, they can be sucked up in a pipette and transferred to the separate dish. If dealing with a large host animal, one with thousands of worms, or several individuals of the same host species, a coning technique can be used to obtain specimens. All of an organ(s) or a sample of the tissue can be placed into a screen cone constructed to fit inside a Pilsner glass containing enough saline to cover the tissue. Periodically, the cone can be shaken gently. After a short period of time, some or all of the worms (of most species) will pass through the screen mesh and end up on the bottom of the glass. Remove the cone and tissue, let the material in the glass settle on the bottom, decant off most of the saline, pour the worms and debris into a Petri dish, and remove the worms. Additional saline may be necessary to find the worms. Place each worm, regardless of whether from the dish or straight from the tissue, into the small dish. Worms from different organs should be kept in separate dishes if there is a later chance of confusion among species and sites. Small worms can be cleaned quickly by flushing them with a finely drawn mouth-pipette. The worm is drawn into the pipette and ejected out forcibly with saline several times. Then any debris should be sucked out and the entire dish of saline replaced so that the cleaned worms remain clean. A Pasteur pipette can be used if the worm is large enough to be constricted to fit into the bore. If the worms are active and like to cling to each other, they should be kept separate in a larger dish. Worms can be separated with 000-00000 red sable spotting brushes without damaging them. Sometimes placing a dissecting pin between the worms and sliding the worms and sliding the worms with a brush up towards the base of the pin (needle) where the gradually increasing diameter will force the worms apart. Fix the worms after they have been cleaned of debris. If there are several specimens, they should be fixed using approximately three different methods. All methods involve heat (or some people prefer glacial acetic acid). The majority of the specimens should be fixed with nearly boiling water or fixative without any pressure. To do this, most or all the saline should be removed from the dish and a relatively large quantity of the hot solution poured into the dish while swirling it. If hot tap water, or other water is used, quickly draw the worms into a pipette and eject them into an empty vial. Remove excess water and replace with 0.5% neutral buffered formalin. Acid fixatives will destroy fine spines. These heat-killed worms will have consistent measurements and, once stained, are good for descriptions and identifications. To be able to view ducts and other special structures, some pressure can be used on specimens in a second method. Place one or more worms of a particular size on a microscope slide with saline and cover with a used or broken coverslip. If you only have two specimens of a single species to prepare using this method, they should be fixed

3 separately to assure one good preparation. The worms are best transferred to the center of the slide using a Pasteur capillary pipette with a nipple and then covered with a coverslip or a piece of a coverslip. Remove enough fluid with the point of a rolled up Kimwipe so that the worm or worms are under slight pressure and straight. If they are bent or twisted and not heavily muscularized, add more saline and start over. Watch the worm under a dissecting scope when removing saline to avoid any damage to the worm. Large or troublesome worms can be relaxed by placing a light bulb near them or by placing them in a refrigerator. The worms can then be fixed by passing the slide over a flame or administering hot water. We have found that heating 30 ml of AFA or 5% buffered formalin over an alcohol lamp in an Erlenmeyer flask until the solution begins to steam works no better than hot water, and that method results in irritation to the person preparing the worms and anyone else in the vicinity . For either method, after fixing the worms, one should immediately add additional fluid so that the worms are not destroyed by the pressure. If you add fluid under the coverslip rather than a flame, raise the coverslip slightly using forceps and a pin on the opposite side (to stabilize the coverslip and avoid smashing the prize specimen) to allow the hot fluid to rapidly fix the worm. Then press down on the coverslip with the pin directly above the worm so that the worm is under a minimum of pressure but is straight, not curled, and fixed (opaque). The slide should be raised from the base of the dissecting scope during this process so that the fixative does not run over onto the glass plate of the scope. The top of a stender dish can be used. If a fixative rather than water is used, let the worms remain under the coverslip for 10 to 30 minutes until they are somewhat hardened. Add fresh fixative if any sign of evaporation is noted. If the worms are fixed by a flame, water should be added immediately, and the worms immediately removed and placed into fixative. Regardless of how the worms are fixed, turnover the coverslip and remove those worms attached to the slide and coverslip and transfer the worms to a vial filled with a small amount (1/5 full) of fixative and a label to identify the host as well as location or site in the host, if necessary to differentiate one group of trematodes from other similar ones. This transfer is best made with a pipette or a 000 brush and never directly with forceps. Enough solution is added so that the coverslip readily slides over the worm(s) without any pressure on it. The coverslip is removed with the forceps in a vertical lift. If one or more worms are attached to the coverslip, turn the coverslip over as indicated above and place it toward one end of the slide. Then add more solution over the worms and either use a pipette (to draw them up or force them) or a brush to place or direct them into the vial containing fixative. To avoid contamination of the pipette or brushes, you can add fixative to the vial with specimens killed with water. After 20 minutes or a few days, depending on the fixative, group of worm, its size, or available time, remove acidic fixatives like AFA (if that is the fixative, and it allows good staining for most species but is destructive to some sclerotized structures) from the tilted vial under a scope and replace it with a full vial of 70% ethyl alcohol. If 5-10% buffered formalin was used, bring the level in the vial up to nearly full. Once stained, these worms fixed under light coverslip pressure allow one to see most structures. If enough specimens are available, one or a few should be fixed under considerable pressure but not enough to cause the worm to burst. The distorted material is not good for measurements, but it will allow an observer to see otherwise covered or twisted ducts and other special features. The combination of the three methods allow for a good understanding of the worms structure and a good description. If an adequate number of specimens are available, some unfixed, un-killed specimens should be added to a vial, from which excess water is removed and exchanged with roomtemperature 95% molecular grade ethanol (or other good solution for future molecular studies). These vials with alcohol-fixed specimens should be stored in the refrigerator.

4 When working with water, saline, and multiple fixatives, separate color-coded pipettes (to avoid later accidental contamination of living worm with fixative) should be used. Hosts are best examined alive to obtain worms in quality condition, but if this can not be done, refrigerate them and examine them within a few hours. Do not freeze hosts or parasites unless you have no choice or if identification (not descriptions) or exact knowledge of sites of large numbers of worms are required (quick freezing)! When laboratory facilities are not available, there are additional methods that can provide good results. Cut a small fish or other animal along the ventral side or make a cut along a piece of intestine, place the material into an empty cup, pour a large volume of boiling water (tap water is fine) quickly over the material (there has to be enough to rapidly fix the entire material), allow debris to settle, decant off water, place material into a ziplock bag (or other container), add fixative of choice (usually 10% formalin), enclose label, and seal bag or other container. Double or triple bag the bags or groups of bags to avoid spillage. Worms can be removed and placed into a vial(s) at a later date. Fixation methods of other groups of metazoan parasites are similar to those for the digeneans with some specific exceptions. Many of those are presented below. Monogeneans There are large and small monogeneans, just like large and small digeneans. They, however, are mostly from fishes and may have hooks, anchors, clamps, suckers, or adhesive pads which make removal from the gills, skin, or other tissue difficult. Often, small species can be obtained in good positions by vigorously shaking the fish, gills, etc. in a jar containing 1 part formalin to 1,000-4,000 water of the type the fish came from (e.g., fresh, brackish, marine). The tissues or fish can then be removed and the solution left alone until the worms settle on the bottom. The water can be decanted, and the worms swirled into a small dish with a mark indicating fixative contamination so that it will not be used later for live specimens. These fixed worms can be transferred to 510% buffered formalin (or some other fixative) or even transferred directly, with no fluid, with a spotting brush to a drop of glycerin or, if a semi-permanent mount is desired, of warmed glycerin jelly placed centrally on a clean slide to be covered as described below. Some of these worms should be saved in a vial of fixative for later staining. Both large and small specimens can also be treated like digeneans, using a slight amount of initial pressure, using a pin and forceps to allow a natural orientation of the haptor and body proper, and using any of several heat-methods to kill and fix the worm. Agents to anesthetize the worms either on or off the host tissue may prove rewarding. Remember that monogeneans live outside the host, so they should be treated with water similar to that which they encounter rather than saline! Cestodes Clean worms in 0.8% saline and then relax them with a lamp, in a refrigerator, with tap or distilled water (not recommended unless necessary for eversion of scolex), by swirling (usually the best single method), or with a combination of these. Usually good preparations can be made by fixing the clean worms with boiling water as they are being swirled in a small amount of saline (so as not to decrease the temperature of the hot killing fluid [near boiling tap water or hot fixative]). The worms are fixed by quickly adding several times as much boiling water or hot fixative to the relaxed worms as the amount of water in which they are being swirled. Remove the worms quickly if killed with hot water (and immediately placed in a vial with buffered formalin or some other

5 fixative. The vial should be large or long enough to place the straightened worm without distorting them. AFA, or the acetic acid in the AFA, or non-buffered formalin will dissolve the calcareous corpuscles (which may be acceptable for some species). In any event, worms fixed in AFA need to be transferred to a vial containing 70% alcohol. If the worms are to be sectioned or nuclear staining is not critical, 5-10% buffered formalin is a better fixer. The tentacles in the scolex, if present, can be extruded either with distilled water or by coverslip pressure. Occasionally, the tentacles will extrude in a hot killing agent. Metacestodes should (at least some individuals) be removed from their cysts or encapsulations, if present, before fixing them. Acanthocephalans The proboscis, which is an important taxonomic character of acanthocephalan, can usually be forced to protrude by placing the worm in distilled water. Use care so that the worm does not become damaged because of swelling. Occasionally some coverslip pressure is necessary to evert the proboscis. Kill the worms in hot water and fix them with buffered formalin or AFA and store them in 70% ethyl alcohol plus 5% glycerin. If you prefer not to kill the worms in hot water, hot fixatives may blister the tegument, in which case a cold fixative should be used. Formalin can be used with good results for many species. It is helpful to prick a few tiny pin holes in the tegument in fixed specimens before beginning staining procedures to allow quicker penetration of fluids into the tissues and to decrease the chances of obtaining a cloudy final product. Nematodes It is best to fix nematodes in concentrated glacial acetic acid for 5-10 minutes, but for most species hot water or hot 70% ethyl alcohol will work. These methods will cause most worms to straighten, an important result for best microscopical observations. Use cold alcohol or 10% formalin for large nematodes if hot methods causes blistering of the cuticle in a test specimen. Remove larval nematodes from encapsulations, if present, before fixing. Store worms in 70% alcohol plus 5% glycerol. Nematodes are generally not stained, except when cotton blue is used to intensify reproductive structures. Adequate examination can be made by gradually clearing worms with glycerin, by quickly clearing them in lactic acid (not toxic to the preparer like lactophenol), or by using a variety of other products and methods. Copepods and Isopods Place these crustaceans directly into 70% alcohol plus 5% glycerin unless they will be sectioned. If they are to be sectioned in situ, they should be fixed with methenamine buffered formalin. Phosphate buffer often leaves a white precipitate on crustaceans. The collector may want to leave fixed parasites attached to gill filaments for later removal or for sectioning. If sections of internal structures are required, Davidsons solution (preferably injected into numerous sites on the cephalothorax region with a fine needled syringe) has produced good results for sectioning the crustaceans. Pathological tissue As a general rule, any tissue to be sectioned should be placed directly into 10% buffered formalin. It is best to leave a parasite attached if other specimens are available as whole mounts. Bouins fixative or Lillies solution, containing formic acid rather than

6 acetic acid, provides a good fixative if bones are present; if other decalcification is necessary, use un-buffered formalin. If immunochemical tests may be conducted, use zinc-formalin. After worms are properly gathered and prepared, they can be examined, stored, stained and mounted, or sent to another for action. Staining and mounting are not necessarily difficult, but it takes time to learn, and experimentation may be necessary. Some of the methods are described below. Whether you are going to do the procedures, have a helper do it, or send them to an expert, a museum, an interested party, or a friend, the time you spend fixing and storing the material will more than pay off for that person in time spent and quality of product produced. The later in the process one waits to clean and orient the specimen, the more difficult and hazardous it becomes to produce a good product. Also, the more information that accompanies the specimen, the more value it will have. This information should include data on the worm, its site in the host, the host identification and identifier, the geographic location, the date, the collector, and a personal host number that can be used to gather any other necessary information from the databook (I suggest a two letter prefix like MS to identify the location, trip, collection, or collector, followed by -07 for year, and -131 for host number). The parasite species or specimen number can follow the host number on the slide at a later date.

PROCEDURE FOR STAINING AND MOUNTING PARASITES I. Small trematodes, acanthocephalans, and miscellaneous parasites Van Cleaves hematoxylin: a. Start with clean specimens. For example, pipette specimens from 70% ethyl alcohol (EtOH) to clean 70% EtOH in a small shell vial or other small container full. If the specimens have not been well cleaned, part of the solution can be withdrawn into a pipette and forcibly squirted back into the vial and on the specimen. This procedure often dislodges debris. Use care not to remove any specimens or splash them out of the vial. If you are uncertain of the location of all the specimens, examine the pipette carefully before using it in another vial. All fluid removed from a vial should be squirted into a separate Stender dish to assure a specimen was not accidentally removed. This dish should be examined for stray worms before discarding the fluid. This entire step should be conducted through a dissecting stereomicroscope. b. Hydrate the specimens in a small container. Add a few drops of distilled water to vial of alcohol or formalin while twirling vial with other hand. Swirling the fluid assures adequate mixing. c. Bring the level of the fluid up to about 4/5 full with 3 to 4 periodic groups of 3-10 drops each. The ultimate goal is to completely replace the alcohol or formalin solution with distilled water. If the specimens are small (about 1

7 mm) and have a thin tegument, the time period between adding drops can be as short as 2 to 3 minutes. The larger the specimens, the longer the period between adding additional water or the fewer the number of drops. For example, thick-tegumented hemiurids or acanthocephalans may take intervals of 1-2 hours. d. When the vial is about 4/5 full (e.g., about 45% alcohol), remove enough fluid so that the level is again about . This should be done under view of a dissecting microscope. Small debris can be removed by pipetting during this step. Continue to slowly bring the level to 4/5 and then remove some fluid until only water is present. The entire hydration process should take about 1 to 1 hours for small worms and 3-24 hours for worms about 1 cm in length. A time of 3 hours is a sufficient period of time for acanthocephalans, if 2 or 3 holes are carefully pricked into the tegument. Use a minutin pin and avoid all internal organs. Hold the worm with a 00000000 (there is often little or no difference among the different unstandardized sizes) red sable brush while inserting the pin. e. Remove all the water except enough to cover the specimens when the vial is held at an angle. Then add some filtered (by filter paper, gauze, or cotton) Van Cleaves hematoxylin. Use less than one pipette full of stain so that the level of the stain is about 1/6 th. It can be diluted, and it is best to let the specimens remain in the stain overnight or, at least, 2 hours. To produce a slightly darker stain quality, you should add 1-2 drops of Ehrlichs hematoxylin to the 2-4 ml of Van Cleaves stain. Cover the shell vial with a 5-ml plastic beaker to prevent evaporation, tipping of the vial, and spilling. One or a few covered shell vials should additionally be covered by a larger beaker or culture dish, so that they are not accidentally dislodged, spilled, or thrown away. f. Remove stain and replace with 1 or 2 exchanges of distilled or deionized water so that the level of clear water is about full. Add 1 to 3 drops of 70% alcohol for 1 to 2 minutes. g. Bring alcohol concentration up to 70% by adding a few drops at a time and removing excess fluid. This should take 45 minutes or more. Both during the hydration and dehydrating processes, the excess water should be pipetted into a small stender dish and continually examined to see if any specimens were accidentally removed. This fluid is discarded into a container as waste. It is imperative when staining several vials of specimens that worms from one vial are not accidentally transferred to a different vial. Each vial should be numbered with waterproof ink and that number should correspond to data that will identify the contained specimens with information such as host identification number and location in the host. It is easiest to keep a log of such data with the number of each type of parasites per vial listed, so if a worm is accidentally transferred to a wrong vial, it can be transferred back to the correct vial. h. Bring the specimens to about 75-80% alcohol and add about 3 to 6 drops of lithium carbonate and let the specimens remain in the solution for about 5 minutes. Then add 2 or enough drops of 0.1M butylamine so that the

8 pH reaches above 7.0. Leave the specimens in the solution for 10 or more minutes. The specimens usually turn brownish in color, which is fine, but if you notice any destaining, remove the solution immediately and replace it with 70% alcohol. If when adding any of the above bases you notice a white precipitate, remove the solution quickly and force the precipitate off with squirts of 70% alcohol or 35% alcohol. The carbonate is not very soluble in alcohol, but it is in water. Nevertheless, the precipitate is difficult to remove from a specimen, so be careful not to add excess lithium carbonate. Adding the bases should prevent future destaining after the mounts are several years old. i. You will now need separate containers of 95% and 100% alcohol. Place only the amount of 100% alcohol that you will use in a container and keep it tightly capped. Take the specimens up to 80 and 85% and then replace with 95% alcohol. Never allow the specimens to come into direct contact with air. They should always be covered with at least a small amount of solution. This precaution should prevent air from getting into the eggs. Replace the 95% with 100% and replace the 100% with a second and perhaps third (if humid) vial of 100% alcohol. Keep the vials covered with the plastic beakers when filled with 100% alcohol. Specimens should remain in each absolute alcohol treatment for about 5 minutes (or less for tiny worms). Contamination of the absolute alcohol with water can be tested by adding a couple of drops into xylene. If the drops produce a cloudy appearance, there is water present, and the alcohol should be replaced. j. As quick as possible to avoid taking up atmospheric water, swirl the solution in the vial so that the specimens are free in the fluid. Transfer the fluid and specimens to a small stender dish. If any of the specimens remain, quickly add more 100% and transfer the worms to the stender dish. k. Remove with pipette all the alcohol from the tipped dish except a minimal amount covering the worms. Quickly pour in 1/5 to dish of clearing agent and keep the worms submerged with a 000 brush. If the worms are very small or difficult to work with, you can add the clearing agent in small amounts to dish of absolute alcohol. This will keep the specimens from rising to the surface. Covering the worms in the stender dish with a coverslip before the clearing agent is added can prevent worms from coming into contact with air. l. After the worms remain submerged by themselves and they are cleared, they are nearly ready to mount. They should be cleaned by brushing any detritus or air bubbles off with a 000 brush. A minutin pin on a wooden applicator can also be helpful. All cleaning must be done under a dissecting scope. Clove oil is a superior clearing agent because it will remove a small amount of water, specimens remain pliable, and specimens can remain in it for days or weeks. Xylene is a poor agent because it will not remove any water, specimens get brittle rapidly, and mounting must be done immediately.

9 m. Clean several slides and coverslips. Set up a working area with a flat cardboard map-form slide holder, a marking pen, a stender dish of xylene, a pair of forceps, a dissecting pin, 2 (000) brushes, Canada balsam or Histoclad (or some other good tested mounting medium; I do not recommend Permount), and a box of Kimwipes. n. Suck up some xylene from a stender dish in a pipette and release it back to the dish before entering the pipette into a container of Canada balsam for the first time. This activity decreases the possibility of air bubbles entering the pipette along with the mounting medium. Pipette a small drop of the medium to the middle of a slide and place the pipette dropper back into the jar of medium after forcing out some of the medium first. Each time medium is added to a slide, the nipple on the pipette should be kept depressed. Excess medium should be squirted along the inside wall of the container before being returned to the medium. This will keep air out of the medium! If you continue this practice, you should have few problems with air bubbles. If bubbles appear on the slide, they can be removed with your brush which should then be dipped into xylene and blotted on a Kimwipe. Usually only one or two bubbles can be removed between each dipping. Xylene will also pop bubbles. If the medium starts to harden at the meniscus before introducing the specimen, add a small amount of xylene to the surface. o. Pick up a specimen with the brush and place it near the middle of the Canada balsam. Clean the clearing agent off the brush with xylene before aligning the specimen. Blot the xylene off the brush by touching it to a Kimwipe. Pick up a coverslip on one edge with the forceps and dip the other side in xylene. Blot off excess xylene so that just a thin film occurs on the coverslip. Now place the dissecting pin on the slide next to the Canada balsam so that the coverslip will not drift and place the side of the coverslip with the most xylene on it next to the pin and slowly lower the coverslip onto the medium. The coverslip should not be grasped but just balanced by the forceps when lowering. The procedure should be done slowly under a dissecting stereomicroscope, and, whenever bubbles appear, the coverslip can be raised up to release the bubbles. If a bubble remains, you may be able to press the coverslip on different sides and cause its movement, forcing the bubble to the edge of the glass. Also, a drop of xylene on the edge of coverslip close to the internal bubble will often cause the bubble to exit. Use care not to dilute the medium too much with xylene. p. Slide, turn, or raise the coverglass slightly using the forceps with the pin as a brace to properly center the specimen. Press the coverslip at a position directly above the specimen very slightly. Do not smash the specimen but put a small amount of pressure on it so that the specimen will not drift. Place the slide in the level tray to prevent drifting or accidents and write a preliminary or final identification number on it. If the specimen is extremely brittle, place a few small shards of broken coverslip in the medium on the slide before adding the specimen. II. Large parasites

10 a. The same procedure indicated above is used but with a Stender dish rather than a shell vial. It may take 1 to 2 days to stain and mount a very large worm. The entire procedure from staining to mounting should take about 1.5 days for 3-8 mm long worms and 2 days for 8-15mm or longer worms. b. Whether the specimens are large or small, only one good specimen should be placed on a slide. Pieces of glass may be necessary to maintain the coverslip approximately parallel with the slide. The preparation should be examined daily and a small amount of medium added to the side of an indentation resulting from evaporation of the xylene in the balsam. Dilute medium may have to be added to one side to avoid trapping an air bubble. Also, a brush bristle or minutin pin may guide a stray bubble from the preparation. III. A. Other stains Delafields and Harris hematoxylin 1. Use the same method as for Van Cleaves hematoxylin until you dehydrate the specimen to 70% alcohol. Destain specimen by adding 0.5% to 1.0% HCl in 70% until the worms are light pink. If the worms are extremely muscular, you can later add 5% HCl for a short period to remove the stain from the tegument without affecting that of internal organs. Wash in 2 to 3 changes of 70% to remove acid and prevent further destaining. Add alkaline 70% alcohol to the specimens until they turn a bluish color. Continue as with other methods.

2. 3. B.

Carmine (Grenachers, Semichons, Mayers and others) 1. Some carmine stains are prepared in 70% alcohol, others in water. If they are prepared in alcohol, they are stained in 70% rather than water, and the initial hydration of the specimens is eliminated. 2. The same procedure is used for destaining. An alkaline step is usually not necessary and sometimes disastrous.

IV.

Stains Some stains are preferable to others for certain species or if certain portions of the anatomy are of interest. Carmine stains cytoplasm and hematoxylin stains nuclei. I prefer Van Cleaves hematoxylin for most but not all trematodes, and I prefer carmine for some cestodes, especially those with hooks. Carmine is not good for seeing all the internal characteristics, but it never destains a specimen as does Van Cleaves if done improperly. Grenachers carmine is probably the

11 best method for acanthocephalans. Harris hematoxylin or Celestin blue are also good for cestodes. Nematodes are not normally stained. V. Mounting and examination of nematodes, arthropods, and others. Glycerin-glycerin jelly: a.Place nematodes or other organisms into a stender or Petri dish full of a solution of 95 parts of 70% ethyl alcohol and 5 parts of glycerin. Let the dish remain either uncovered for several days to allow the alcohol to evaporate or covered with a Kimwipe or gauze to prevent the settling of dust. The glycerin slowly clears the nematodes. The period of time can be reduced by placing them in an oven at 37-42 C. Higher temperatures will probably harden the worms. Directly transferring the worms from alcohol to glycerin would disrupt them. Nematodes can be stored in glycerin, glycerin and a little formalin, or 70% alcohol plus 5 parts glycerin. They are best studied by leaving them in glycerin and rotating them under a coverslip so that all sides of the worm can be observed. After observations are made, the worms are transferred back to a vial of glycerin. They can also be cleared and examined easily by taking them directly from alcohol to lactic acid or lactophenol (less desirable because of toxic fumes) and replacing them directly into alcohol when finished. b. Worms left uncovered in glycerin provide a tasty morsel for a cockroach or other pest, so care to protect valuable specimens must be attempted. c.When semi-permanent mounts are desired, the worms can be transferred directly from glycerin to the glycerin jelly, but the jelly must be melted. Melting is best accomplished by placing a small container of jelly in a culture dish or Petri dish of water on a hot plate. When the jelly has melted, suck up a large amount of it in a clean pipette and leave the pipette in the container. Be sure not to introduce air bubbles into the pipette. This is easily done by sucking up some warm water into the pipette and expelling it on the floor or sink before introducing the pipette into the glycerin for the first time. d.Place a small amount of melted jelly in the middle of a clean slide and carefully remove any air bubbles with a brush. The brush can be cleaned between dips by placing it in hot water and blotting it on a Kimwipe. Transfer a single worm or a male and female pair with the brush to the jelly, clean the brush in water, and center the worms. If the jelly begins to harden, place the slide directly on the hot plate. Place a dissecting pin on one side of the jelly and place a clean coverslip slowly over the jelly using the pin as a stop. Breathing on a coverslip will add a thin film of condensation, which will help prevent air bubbles. The whole process is carried out under a dissecting scope. e.The jelly will harden within a few minutes. If you wish to quicken the hardening process, place the slide onto a piece of ice. After a few days the excess jelly can be removed with a razor blade and a damp Kimwipe. The preparation should then be ringed using acrylic or enamel

12 paint or a special cement. This is better than fingernail polish. This material will prevent air bubbles from entering or the coverslip from coming off on a warm day. These are not permanent mounts and the worms can be easily removed by cutting the ringing compound and warming the slide. PARASITOLOGY - OVERSTREET Formulae of useful fixatives, stains, and mounting media PVA Fixative Mercuric chloride sat. aq. sol. Glycerin Glacial acetic acid Ethyl alcohol, 95% Powdered PVA (Evannai 90-25 or 71-24) Schaudinns Fixative (for protozoa) Mercuric chloride, sat. aq. sol. Ethyl alcohol, 95% Glacial acetic acid (add just before use) AFA Fixative Ethyl alcohol, 95% Formaldehyde (37-40% sol.) Glacial acetic acid Distilled water Davidsons Fixative (for crustaceans) Ethyl alcohol, 95% Formaldehyde (40% sol.) Glacial acetic acid Distilled (or tap) water Bouins Fixative Picric acid, sat. aq. sol. Formaldehyde (40% sol.) Glacial acetic acid Lillies Fluid (to decalcify small bones) Picric acid, sat. aq. sol. (1.2%) Formaldehyde (40% sol.) Formic acid (90-95% aqueous) 10% Formalin 85 10 5 ml ml ml 75 25 5 ml ml ml 33 22 11.5 33.5 ml ml ml ml 50 10 5 45 ml ml ml ml 200 ml 100 ml 15 ml 312 7.5 25 156 25 ml ml ml ml ml

13 Formaldehyde (40% sol.) Distilled water 10% Formalin: buffered Formaldehyde Na2H2PO4 Dibasic NaH2PO4 Monobasic Distilled water Alcohol and Glycerin Preservative Ethyl alcohol, 70% Glycerin Lugols Iodine Potassium iodide Iodine (powdered crystals) Distilled water 10 5 100 gm gm ml 95 5 ml ml 10 90 10 0.65 0.35 90 ml ml ml gm gm ml

Heidenhains Iron Alum-Hematoxylin (for fecal smears) Mordant: Distilled water Ferric ammonium sulfate (clear lavender crystals) Stain: Distilled water Hematoxylin dissolved in 10ml Ethyl alcohol, 95% 100 0.5 ml gm 100 2 ml gm

Stain must be aged or artificially aged and mordant should be prepared just before use. Ehrlichs Hematoxylin Hematoxylin Ethyl alcohol, 95% Glycerin Distilled water Glacial acetic acid Potassium alum (K2SO4 Al2(SO4)324H20) 2 100 100 100 10 3 gm ml ml ml ml gm

Ripen for several weeks or ripen artificially with addition of 0.2gm sodium iodate. Delafields Hematoxylin Hematoxylin Ethyl alcohol, 95% Aluminum ammonium sulfate, sat. aq. sol. 4 25 400 gm ml ml

14 Glycerin 100 ml Methyl alcohol 100 ml Dissolve hematoxylin in ethyl alcohol, add aluminum alum solution and let stand one week exposed to air and light. Filter and then add glycerin and methyl alcohol. Let stand 6 8 weeks. Van Cleaves Hematoxylin Delafields hematoxylin Ehrlichs hematoxylin Aluminum ammonium sulfate Distilled water Usually takes several months to ripen. Semichons Aceto-carmine Glacial acetic acid Distilled water Carmine (alum lake-certified) 100 100 as desired ml ml 1 1 6.5 100 ml ml gm ml

Heat solution to 95C for 15 minutes in reflux apparatus, cool, and filter. Mayers HCl- carmine Ethyl alcohol, 95% Distilled water Concentrated hydrochloric acid Carmine 90 15 1 4 ml ml ml gm

Mix carmine, water, and acid and boil until dye is dissolved. Add 95% alcohol, filter, and neutralize with ammonia and re-filter. Gallocyanin-Chrome alum Chromium potassium sulfate Distilled water Gallocyanin 5 100 0.15 gm ml gm

Dissolve chrome alum in water, add gallocyanin and boil for 5 minutes. Cool, filter, adjust to 100 ml and adjust pH to 0.83. Note: you can add chrome alum and gallocyanin to the water, bring to a boil and simmer for 20 minutes. Cool and filter. Chrome alum is chromium potassium sulphate dodecahydrate, CrK(SO4)212H2O. The intial solution pH is ~1.64 and changing this will either eliminate or accentuate nonspecific staining. Addition of up to 10ml N hydrochloric acid will eliminated background staining. Glycerin jelly (with phenol) Gelatin Distilled water 10 60 gm ml

15 Glycerin Phenol (melted crystals)* *(to eliminate contamination with microorganisms) Glycerin jelly (with chrom-alum) Gelatin Glycerin Potassium chromium sulfate Distilled water Hoyers Berlese Mounting Media for Small Arthropods Distilled water Gum Arabic (clear crystals) Chloral hydrate Glycerin Lithium carbonate Saturated solution of Lithium carbonate in final ~ 70-85% ethyl alcohol (EtOH) solution. Place lithium carbonate into 50% EtOH to dissolve (solution will be cloudy), let sit for 1224 hours then add enough 95% EtOH to the solution to bring it up to desired EtOH concentration. Butylamine 0.1M butylamine in distilled water = 0.1M) (73.137g/L = 1M therefore, 7.3137g/1000ml 50 30 200 20 ml gm gm ml 3 20 0.2 80 gm ml gm ml 70 0.5 ml ml

Methenamine (Hexamine) Buffered Formalin Hexamine 40% formalin 200 gm 1000 ml